A γ-secretase inhibitor, but not a γ-secretase modulator, induced defects in BDNF axonal trafficking and signaling: evidence for a role for APP.

Clues to Alzheimer disease (AD) pathogenesis come from a variety of different sources including studies of clinical and neuropathological features, biomarkers, genomics and animal and cellular models. An important role for amyloid precursor protein (APP) and its processing has emerged and considerable interest has been directed at the hypothesis that Aβ peptides induce changes central to pathogenesis. Accordingly, molecules that reduce the levels of Aβ peptides have been discovered such as γ-secretase inhibitors (GSIs) and modulators (GSMs). GSIs and GSMs reduce Aβ levels through very different mechanisms. However, GSIs, but not GSMs, markedly increase the levels of APP CTFs that are increasingly viewed as disrupting neuronal function. Here, we evaluated the effects of GSIs and GSMs on a number of neuronal phenotypes possibly relevant to their use in treatment of AD. We report that GSI disrupted retrograde axonal trafficking of brain-derived neurotrophic factor (BDNF), suppressed BDNF-induced downstream signaling pathways and induced changes in the distribution within neuronal processes of mitochondria and synaptic vesicles. In contrast, treatment with a novel class of GSMs had no significant effect on these measures. Since knockdown of APP by specific siRNA prevented GSI-induced changes in BDNF axonal trafficking and signaling, we concluded that GSI effects on APP processing were responsible, at least in part, for BDNF trafficking and signaling deficits. Our findings argue that with respect to anti-amyloid treatments, even an APP-specific GSI may have deleterious effects and GSMs may serve as a better alternative

Loss of Caveolin-1 Accelerates Neurodegeneration and Aging

The aged brain exhibits a loss in gray matter and a decrease in spines and synaptic densities that may represent a sequela for neurodegenerative diseases such as Alzheimer's. Membrane/lipid rafts (MLR), discrete regions of the plasmalemma enriched in cholesterol, glycosphingolipids, and sphingomyelin, are essential for the development and stabilization of synapses. Caveolin-1 (Cav-1), a cholesterol binding protein organizes synaptic signaling components within MLR. It is unknown whether loss of synapses is dependent on an age-related loss of Cav-1 expression and whether this has implications for neurodegenerative diseases such as Alzheimer's disease.We analyzed brains from young (Yg, 3-6 months), middle age (Md, 12 months), aged (Ag, >18 months), and young Cav-1 KO mice and show that localization of PSD-95, NR2A, NR2B, TrkBR, AMPAR, and Cav-1 to MLR is decreased in aged hippocampi. Young Cav-1 KO mice showed signs of premature neuronal aging and degeneration. Hippocampi synaptosomes from Cav-1 KO mice showed reduced PSD-95, NR2A, NR2B, and Cav-1, an inability to be protected against cerebral ischemia-reperfusion injury compared to young WT mice, increased Aβ, P-Tau, and astrogliosis, decreased cerebrovascular volume compared to young WT mice. As with aged hippocampi, Cav-1 KO brains showed significantly reduced synapses. Neuron-targeted re-expression of Cav-1 in Cav-1 KO neurons in vitro decreased Aβ expression.Therefore, Cav-1 represents a novel control point for healthy neuronal aging and loss of Cav-1 represents a non-mutational model for Alzheimer's disease

Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are increasingly viewed as an early feature of neurodegenerative diseases, including Alzheimer’s disease (AD) and Huntington’s disease (HD). As yet unclear is the mechanism(s) by which axonal injury is induced. We reported the development of a novel technique to produce biologically active, monobiotinylated BDNF (mBtBDNF) that can be used to trace axonal transport of BDNF. Quantum dot-labeled BDNF (QD-BDNF) was produced by conjugating quantum dot 655 to mBtBDNF. A microfluidic device was used to isolate axons from neuron cell bodies. Addition of QD-BDNF to the axonal compartment allowed live imaging of BDNF transport in axons. We demonstrated that QD-BDNF moved essentially exclusively retrogradely, with very few pauses, at a moving velocity of around 1.06 μm/sec. This system can be used to investigate mechanisms of disrupted axonal function in AD or HD, as well as other degenerative disorders

Targeting increased levels of APP in Down syndrome: Posiphen-mediated reductions in APP and its products reverse endosomal phenotypes in the Ts65Dn mouse model.

ObjectiveRecent clinical trials targeting amyloid beta (Aβ) and tau in Alzheimer's disease (AD) have yet to demonstrate efficacy. Reviewing the hypotheses for AD pathogenesis and defining possible links between them may enhance insights into both upstream initiating events and downstream mechanisms, thereby promoting discovery of novel treatments. Evidence that in Down syndrome (DS), a population markedly predisposed to develop early onset AD, increased APP gene dose is necessary for both AD neuropathology and dementia points to normalization of the levels of the amyloid precursor protein (APP) and its products as a route to further define AD pathogenesis and discovering novel treatments.BackgroundAD and DS share several characteristic manifestations. DS is caused by trisomy of whole or part of chromosome 21; this chromosome contains about 233 protein-coding genes, including APP. Recent evidence points to a defining role for increased expression of the gene for APP and for its 99 amino acid C-terminal fragment (C99, also known as β-CTF) in dysregulating the endosomal/lysosomal system. The latter is critical for normal cellular function and in neurons for transmitting neurotrophic signals.New/updated hypothesisWe hypothesize that the increase in APP gene dose in DS initiates a process in which increased levels of full-length APP (fl-APP) and its products, including β-CTF and possibly Aβ peptides (Aβ42 and Aβ40), drive AD pathogenesis through an endosome-dependent mechanism(s), which compromises transport of neurotrophic signals. To test this hypothesis, we carried out studies in the Ts65Dn mouse model of DS and examined the effects of Posiphen, an orally available small molecule shown in prior studies to reduce fl-APP. In vitro, Posiphen lowered fl-APP and its C-terminal fragments, reversed Rab5 hyperactivation and early endosome enlargement, and restored retrograde transport of neurotrophin signaling. In vivo, Posiphen treatment (50 mg/kg/d, 26 days, intraperitoneal [i.p.]) of Ts65Dn mice was well tolerated and demonstrated no adverse effects in behavior. Treatment resulted in normalization of the levels of fl-APP, C-terminal fragments and small reductions in Aβ species, restoration to normal levels of Rab5 activity, reduced phosphorylated tau (p-tau), and reversed deficits in TrkB (tropomyosin receptor kinase B) activation and in the Akt (protein kinase B [PKB]), ERK (extracellular signal-regulated kinase), and CREB (cAMP response element-binding protein) signaling pathways. Remarkably, Posiphen treatment also restored the level of choline acetyltransferase protein to 2N levels. These findings support the APP gene dose hypothesis, point to the need for additional studies to explore the mechanisms by which increased APP gene expression acts to increase the risk for AD in DS, and to possible utility of treatments to normalize the levels of APP and its products for preventing AD in those with DS.Major challenges for the hypothesisImportant unanswered questions are: (1) When should one intervene in those with DS; (2) would an APP-based strategy have untoward consequences on possible adaptive changes induced by chronically increased APP gene dose; (3) do other genes present on chromosome 21, or on other chromosomes whose expression is dysregulated in DS, contribute to AD pathogenesis; and (4) can one model strategies that combine the use of an APP-based treatment with those directed at other AD phenotypes including p-tau and inflammation.Linkage to other major theoriesThe APP gene dose hypothesis interfaces with the amyloid cascade hypothesis of AD as well as with the genetic and cell biological observations that support it. Moreover, upregulation of fl-APP protein and products may drive downstream events that dysregulate tau homeostasis and inflammatory responses that contribute to propagation of AD pathogenesis

A γ-secretase inhibitor, but not a γ-secretase modulator, induced defects in BDNF axonal trafficking and signaling: evidence for a role for APP.

Clues to Alzheimer disease (AD) pathogenesis come from a variety of different sources including studies of clinical and neuropathological features, biomarkers, genomics and animal and cellular models. An important role for amyloid precursor protein (APP) and its processing has emerged and considerable interest has been directed at the hypothesis that Aβ peptides induce changes central to pathogenesis. Accordingly, molecules that reduce the levels of Aβ peptides have been discovered such as γ-secretase inhibitors (GSIs) and modulators (GSMs). GSIs and GSMs reduce Aβ levels through very different mechanisms. However, GSIs, but not GSMs, markedly increase the levels of APP CTFs that are increasingly viewed as disrupting neuronal function. Here, we evaluated the effects of GSIs and GSMs on a number of neuronal phenotypes possibly relevant to their use in treatment of AD. We report that GSI disrupted retrograde axonal trafficking of brain-derived neurotrophic factor (BDNF), suppressed BDNF-induced downstream signaling pathways and induced changes in the distribution within neuronal processes of mitochondria and synaptic vesicles. In contrast, treatment with a novel class of GSMs had no significant effect on these measures. Since knockdown of APP by specific siRNA prevented GSI-induced changes in BDNF axonal trafficking and signaling, we concluded that GSI effects on APP processing were responsible, at least in part, for BDNF trafficking and signaling deficits. Our findings argue that with respect to anti-amyloid treatments, even an APP-specific GSI may have deleterious effects and GSMs may serve as a better alternative

Standards for preclinical research and publications in developmental anaesthetic neurotoxicity: expert opinion statement from the SmartTots preclinical working group

In March 2019, SmartTots, a public-private partnership between the US Food and Drug Administration and the International Anesthesia Research Society, hosted a meeting attended by research experts, anaesthesia journal editors, and government agency representatives to discuss the continued need for rigorous preclinical research and the importance of establishing reporting standards for the field of anaesthetic perinatal neurotoxicity. This group affirmed the importance of preclinical research in the field, and welcomed novel and mechanistic approaches to answer some of the field's largest questions. The attendees concluded that summarising the benefits and disadvantages of specific model systems, and providing guidance for reporting results, would be helpful for designing new experiments and interpreting results across laboratories. This expert opinion report is a summary of these discussions, and includes a focused review of current animal models and reporting standards for the field of perinatal anaesthetic neurotoxicity. This will serve as a practical guide and road map for novel and rigorous experimental work

Swedish Nerve Growth Factor Mutation (NGFR100W) Defines a Role for TrkA and p75NTR in Nociception

Nerve growth factor (NGF) exerts multiple functions on target neurons throughout development. The recent discovery of a point mutation leading to a change from arginine to tryptophan at residue 100 in the mature NGFβ sequence (NGFR100W) in patients with hereditary sensory and autonomic neuropathy type V (HSAN V) made it possible to distinguish the signaling mechanisms that lead to two functionally different outcomes of NGF: trophic versus nociceptive. We performed extensive biochemical, cellular, and live-imaging experiments to examine the binding and signaling properties of NGFR100W Our results show that, similar to the wild-type NGF (wtNGF), the naturally occurring NGFR100W mutant was capable of binding to and activating the TrkA receptor and its downstream signaling pathways to support neuronal survival and differentiation. However, NGFR100W failed to bind and stimulate the 75 kDa neurotrophic factor receptor (p75NTR)-mediated signaling cascades (i.e., the RhoA-Cofilin pathway). Intraplantar injection of NGFR100W into adult rats induced neither TrkA-mediated thermal nor mechanical acute hyperalgesia, but retained the ability to induce chronic hyperalgesia based on agonism for TrkA signaling. Together, our studies provide evidence that NGFR100W retains trophic support capability through TrkA and one aspect of its nociceptive signaling, but fails to engage p75NTR signaling pathways. Our findings suggest that wtNGF acts via TrkA to regulate the delayed priming of nociceptive responses. The integration of both TrkA and p75NTR signaling thus appears to regulate neuroplastic effects of NGF in peripheral nociception.SIGNIFICANCE STATEMENT In the present study, we characterized the naturally occurring nerve growth factor NGFR100W mutant that is associated with hereditary sensory and autonomic neuropathy type V. We have demonstrated for the first time that NGFR100W retains trophic support capability through TrkA, but fails to engage p75NTR signaling pathways. Furthermore, after intraplantar injection into adult rats, NGFR100W induced neither thermal nor mechanical acute hyperalgesia, but retained the ability to induce chronic hyperalgesia. We have also provided evidence that the integration of both TrkA- and p75NTR-mediated signaling appears to regulate neuroplastic effects of NGF in peripheral nociception. Our study with NGFR100W suggests that it is possible to uncouple trophic effect from nociceptive function, both induced by wild-type NGF

Differential effects of BMS-299897 and sGSM41 on APP processing.

<p><b>A</b>: A diagram depicts APP processing and the pathways that GSI or GSM treatment differentially affects Aβ peptide formation and the production of APP C-terminal fragments (APP CTFs). First, β-secretase or α-secretase cleaves APP, leading to the production of either β-CTF or α-CTF. Cleavage of β-CTF by γ-secretase at multiple sites yields several Aβ peptides and the APP intracellular domain (AICD). Cleavage of α-CTF by γ-secretase gives rise to and AICD and the P3 fragment. <b>B</b>: Differential effects of GSI and GSM on the production of Aβ species and APP β-CTF [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118379#pone.0118379.ref034" target="_blank">34</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118379#pone.0118379.ref036" target="_blank">36</a>]. Rat E18 cortical neurons (DIV7) were treated with GSI BMS-299897 (<b>C</b>) or sGSM41 (<b>D</b>) for 24 hrs. The media were harvested and levels of Aβ species (Aβ38, 40, 42) in the media were measured as described in Materials and Methods (n = 3, *P < 0.05, **P < 0.01 using student’s <i>t</i>-test). Treatment with 1μM BMS-299897 or 2.5μM sGSM41 showed the most robust effect and these conditions were used in all other experiments herein. <b>E</b>: Western blotting analyses showing the processing of two substrates of γ-secretase, APP and N-cadherin, in cortical neurons treated with vehicle (0.1% DMSO), 1μM BMS-299897, 2.5μM sGSM41 for 24 hrs. <b>F</b>: Quantitative measurement of mRNA levels by real-time PCR in cortical neurons treated as in <b>E</b>. The mRNA levels are normalized to mRNA levels of β-actin (n = 3, mean±S.E., p values represent results of Student’s <i>t</i>-test).</p

BMS-299897, not sGSM41, alters the distribution of mitochondria and synaptophysin-positive vesicles within processes of rat E18 hippocampal neurons.

<p><b>A</b>: Experimental design for examining distribution of mitochondria and synaptic vesicle precursors within neuronal processes. Rat E18 hippocampal neurons at DIV4 were treated every 24 hrs with vehicle, BMS-299897 or sGSM41. Neurons at DIV7 were either labeled with MitoTracker for analysis of mitochondria (<b>B-D</b>) or fixed and stained for synaptophysin with a specific antibody (<b>E-G</b>). <b>B</b>: Representative images of DIC and MitoTracker under each treatment conditions. The density of mitochondria (<b>C</b>) and the measurement of mitochondrial profile (<b>D</b>) are quantitated and presented (n = 15–20 neurites for vehicle, sGSM41 samples. For BMS-299897-treated samples, n = 95–150 puncta, mean±S.E., ***P<0.001). <b>E</b>: Representative images of DIC and synaptophysin immmunostaining for each treatment condition. The density of mitochondria (<b>F</b>) and the measurement of mitochondrial profile (<b>G</b>) are quantitated and presented (For density analysis, n = 29–40 neurites, mean±S.E., *P<0.05. For profile measurement, n = 26–27 puncta). Scale bar = 20μm.</p

Knockdown of APP rescues deficits in velocity and directionality of axonally transported QD-BDNF induced by BMS-299897.

<p><b>A</b>: Western blotting analysis of typical siRNA-mediated knockdown effect on full length APP and APP CTFs. Rat E18 hippocampal neurons (DIV5) were transfected with siRNA against APP or control siRNA for 72 hrs followed by drug treatment for 24 hrs. On average, the protein levels of APP and APP CTF were knocked down to 70% of normal levels. <b>B</b>: Live imaging of axonal transport of QD-BDNF was performed as in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118379#pone.0118379.g002" target="_blank">Fig. 2</a></b>and representative kymographs of QD-BDNF movement (60 sec) within axons are shown for each condition. Retrograde direction is indicated. Analysis of QD-BDNF for average velocities (<b>C</b>) and directionality of QD-BDNF movement (<b>D</b>) revealed that knocking down APP prior to BMS-299897 treatment partially rescued the deficits in both the velocities and directionality of QD-BDNF as seen previously. 15–20 separate movies were collected and analyzed for each chamber. The data represent 50–130 QD-BDNF molecules (mean±S.E.). Neurons that were cultured in microfluidic chambers were transfected with siRNA and treated with BMS-299897 as in <b>A</b>. BDNF (50 ng/ml, final concentration) was added to the distal axon chambers only for 30 min. Neurons were fixed and stained for pCREB using a specific antibody. The nuclei were stained with the Hoechst dye. Representative images are shown in <b>E</b> and quantitative analysis of the percentage of nuclei that were pCREB-positive is shown in <b>F</b> (mean±S.E., n = 10 images, **P = 0.004). All images were obtained on a Leica DMI6000B inverted microscope (scale bar = 50μm).</p